Hearing impairment is often caused by the sudden or gradual death of the cochlear hair cells, which are vulnerable to damage from noise, age, infection and toxic drugs. Mammals are unable to regenerate lost hair cells resulting in permanent and irreversible hearing ability. A predominant mechanism of hair cell death is via apoptosis in response to reactive oxygen species (ROS) caused by toxic drugs or noise. Defining cell death mechanisms and the time course of their action within hair cell apoptosis would provide targets for pharmacological intervention that could block or reverse hair cell damage. The objective of this research is to understand the role of mitochondrial function in mammalian cochlear hair cell apoptosis in vivo. Mitochondria have been well-established to be the powerhouse of the cell by generating most of cells' ATP, but these organelles also have a role in detoxifying the cell from reactive ROS accumulation, regulating Ca[+2] homeostasis, and initiating apoptosis in response to injury. In the presence of a toxic substance, cytochrome c is released from the space between the mitocondrion's inner and outer membrane, which once in the cytosol, activate caspases that destroy the cell. Due to the high energy demands of cochlear hair cells, they are particularly vulnerable to damage from mitochondrial ROS accumulation. Furthermore, outer hair cells engage in prestin-mediated cell motility suggesting their energy demands are higher than inner hair cells, providing a hypothesis as to why they are nearly always lost first during ototoxic stress. To define the role of ototoxic mitochondrial dysfunction in hair cell apoptosis, I will use fluorescence microscopy techniques in adult guinea pigs in vivo and in postnatal rat cochlear cultures in vitro. I will use potentometric and targeted biomarker dyes to directly measure changes in mitochondrial membrane potential, in the accumulation of mitochondrial ROS, in levels of reduced glutathione (GSH, an indicator of oxidative stress) and in plasma membrane integrity. The ability to image cellular changes in hair cells occurring as a result of ototoxicity would be a tremendous advance for basic researchers seeking to correlate cellular characteristics of the living cochlea with real-time imaging with fluorescent biomarkers of cell function. Thus, this work could fundamentally affect the way that hearing research is conducted by allowing longitudinal investigations into the intact mammalian cochlea for the first time.